Epigallocatechin dimers or trimers having lipase inhibitory activity and/or antioxidant activity

ABSTRACT

An object of the present invention is to provide a lipase activity inhibitor that shows high inhibitory activity against pancreatic lipase to suppress the absorption of meal-derived fat and/or which contributes to suppressing and preventing obesity, as well as a food or beverage that has such lipase activity inhibitor incorporated therein. Another object of the invention is to provide a lipase inhibitor of tea origin that suits most consumers&#39; taste and which will not impair the flavor of the food or beverage when incorporated therein. Still another object of the invention is to provide a process for producing said lipase inhibitors. Further object of the invention is to provide antioxidants. To attain these objects, epigallocatechin dimers (oolong homobisflavans) or trimers are incorporated in foods or beverages. As a result, the absorption of meal-derived fat can be suppressed and, in addition, antioxidation effect is obtained. The compounds of the invention can be produced by reacting epigallocatechin gallate with formaldehyde in the presence of an acid.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. National Stage Application of InternationalApplication No. PCT/JP2005/009666, filed May 26, 2005, and claimsbenefit of Japanese application No. 158462-2004 filed May 27, 2004.

TECHNICAL FIELD

This invention relates to epigallocatechin dimers or trimers havinglipase inhibitory activity, and foods and beverages that have thosedimers or trimers incorporated therein. The invention also relates toepigallocatechin dimers or trimers having antioxidant action, and foodsand beverages that have those dimers or trimers incorporated therein.The invention further relates to processes for producing thoseepigallocatechin dimers and trimers.

BACKGROUND ART

Lipase Inhibitor

With the recent westernization of life style in Japan, the intake ofhigh-fat meals by the Japanese people is ever increasing. According tothe 1999 Japanese national nutrition survey, the energy intake of theJapanese people has been decreasing each year but yet their lipid energyratio has exceeded the normal levels of about 25%. Also, 50-60% ofpeople aged over 60 have been found to have higher-than-normal neutralfat and cholesterol levels (Ministry of Health, Labor and Welfare, “1999National Nutrition Survey Results Summarized”, Rinshou Eiyou 2001,98(5)577-588).

Obesity is one of the most serious conditions in modern society and ismainly ascribed to excessive intake of lipids. Besides obesity,excessive lipid intake is known to cause the onset of associateddiseases including diabetes, hyperlipemia, hypertension, andarteriosclerosis. The only drug that has been approved in Japan fortreating obesity is the appetite suppressant Mazindol (registeredtrademark) but Mazindol has been reported to cause side effects such asdry mouth, constipation, discomfort in the stomach, vomiturition ornausea, etc. (Rinsho Hyoka, 1985, 13(2), pp. 419-459; Rinsho Hyoka,1985, 13(2), pp. 461-515). Outside Japan, Zenical (registeredtrademark), a drug having sufficient lipase inhibitory activity tosuppress intestinal fat absorption, is on the market for treatingobesity, but Zenical has also been reported to cause various sideeffects such as fatty stools, frequent bowel movements, loose passage,diarrhea, stomachache, etc. (Lancet, 1998, 352, pp. 67-172).

Another way that is known to be effective in preventing obesity is toreduce caloric intake through a diet regimen. However, this approachrequires strict nutritional guidance and control and, hence, isdifficult to implement in everyday life. Therefore, if body absorptionof meal-derived fat could be suppressed in a safe and healthy manner, apractical and effective measure could be offered for treating obesityand associated diseases and promoting health.

Under the circumstances, attention is being drawn to the development of“foods for specified health uses” which have been proven to be safe andeffective in humans. Foods for specified health uses that have been soldto date as having the ability to suppress the increase of postprandialserum neutral fat levels include a globin digest that suppresses fatabsorption by inhibiting pancreatic lipase (J. Nutr. 1988, 128, pp.56-60; Journal of Nutritional Science and Vitaminology, 1999, 52(2), pp.71-77; Kenkou•Eiyou Shokuhin Kenkyu, Japan Health Food & Nutrition FoodAssociation, 2002, 5(3), pp. 131-144), diacyl glycerol having differentdigestion/absorption characteristics compared to triacyl glycerol (J.Am. Coll. Nutr. 2000, 19(6), pp. 789-796; Clin. Chim. Acta. 2001, 11(2),pp. 109-117), and eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA) that are purified from fish oil. In order to suppress theabsorption of dietary fat using the foods for specified health useslisted above, it is desirable that they are ingested together withmeals. However, it is predicted that the foods listed above may affectthe flavor of food and beverage to be taken in with the above-listedfoods.

Some polyphenols are known to have activity in inhibiting lipase. Amongthe examples reported so far are tannin derived from plant bark (JP60-11912 B), tannins or flavonoids and glycosides thereof that arecontained in the leguminous plant Cassia nomame (JP 8-259557 A), lipidabsorption suppressing foods having incorporated thereinepigallocatechin gallate and epicathechin gallate that are the maincomponents in green tea (JP 3-228664 A), lipase inhibitors comprisingwater extracts of green pepper, shimeji mushroom, pumpkin, Grifolafrondosa, Sargassum fusiforme (Harvey) Setchell, green tea or oolong tea(JP 3-219872 A), flavone and flavonols (JP 7-61927 A), hydroxybenzoicacids (gallic acid) (JP 1-102022 A), triterpene compounds and theirderivatives (JP 9-40689 A), and antiobesity drugs containing procyanidinof tamarind as an active ingredient (JP 9-291039 A). Also known arelipase inhibitory action of grape seed extract (Nutrition, 2003, 19,(10), pp. 876-879), the lipase inhibitory action of Salacia oblongaderived polyphenols and their antiobesity action in rat (J. Nutr., 2002,132, pp. 1819-1824) and the antiobesity action of oolong tea extract inmouse (Int. J. Obes., 1999, 23, pp. 98-105).

The lipid lowering effect of oolong tea has been reported by manyresearchers. In one report, subjects were allowed to drink commercialoolong tea in 1330 ml portions daily for six weeks to show a significantdrop in blood neutral fat levels (Journal of Nutritional Science andVitaminology, 1991, 44(4), pp. 251-259) and in another, 102 male andfemale subjects with simple obesity were orally administered oolong tea(2 g×4/day) for six consecutive weeks and a weight loss of at least 1 kgwas observed in 67% of the subjects and, in addition, the subjectsshowing high blood neutral fat levels exhibited significant improvementsafter ingesting oolong tea (Journal of The Japanese Society of ClinicalNutrition, 1998, 20(1), pp. 83-90).

-   Non-patent document 1: Ministry of Health, Labor and Welfare, “1999    National Nutrition Survey Results Summarized”, Rinshou Eiyou 2001,    98(5)577-588-   Non-patent document 2: Rinsho Hyoka, 1985, 13(2), pp. 419-459-   Non-patent document 3: Rinsho Hyoka, 1985, 13(2), pp. 461-515-   Non-patent document 4: Lancet, 1998, 352, pp. 67-172-   Non-patent document 5: J. Nutr. 1988, 128, pp. 56-60-   Non-patent document 6: Journal of Nutritional Science and    Vitaminology, 1999, 52(2), pp. 71-77-   Non-patent document 7: Kenkou•Eiyou Shokuhin Kenkyu, Japan Health    Food & Nutrition Food Association, 2002, 5(3), pp. 131-144-   Non-patent document 8: J. Am. Coll. Nutr. 2000, 19(6), pp. 789-796-   Non-patent document 9: Clin. Chim. Acta. 2001, 11(2), pp. 876-879-   Non-patent document 10: Nutrition, 2003, 19, (10), pp.-   Non-patent document 11: J. Nutr., 2002, 132, pp. 1819-1824-   Non-patent document 12: Int. J. Obes., 1999, 23, pp. 98-105-   Non-patent document 13: Journal of Nutritional Science and    Vitaminology, 1991, 44(4), pp. 251-259-   Non-patent document 14: Journal of The Japanese Society of Clinical    Nutrition, 1998, 20(1), pp. 83-90-   Patent document 1: JP 60-11912 B-   Patent document 2: JP 8-259557 A-   Patent document 3: JP 3-228664 A-   Patent document 4: JP 3-219872 A-   Patent document 5: JP 7-61927 A-   Patent document 6: JP 1-102022 A-   Patent document 7: JP 9-40689 A-   Patent document 8: JP 9-291039 A

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The previously reported lipase inhibitors shown above are by no meanssatisfactory in their efficacy. Even if the extract of a certain plantis shown to be effective, its lipase inhibitory activity is difficult tomaintain in a consistent way unless the amount of the active ingredientin the plant extract is clarified, because the extract is of naturalorigin.

In addition, a lipase inhibitor derived from a plant that does not suitmany consumers' taste is most likely to affect the flavor of food orbeverage in which it is incorporated. Hence, lipase inhibitorsoriginating from tea that suits many consumers' taste can be promisingcandidates. On the other hand, if one wants to lower the lipid levelwith oolong tea that suits many consumers' taste, it has to be drunk inlarge enough quantities to prove effective but this is not practical ineveryday life. Supplying a simple concentrate of oolong tea is not apractical method, either, since it has too much bitterness and pungencyand also high caffeine content.

An object, therefore, of the present invention is to provide a lipaseactivity inhibitor that shows high inhibitory activity againstpancreatic lipase to suppress the absorption of meal-derivedtriglyceride and/or which contributes to suppressing and preventingobesity.

Another object of the present invention is to provide a lipase inhibitorof oolong tea origin that suits many consumers' taste and which will notimpair the flavor of the food or beverage when incorporated therein.

Still another object of the present invention is to provide processesfor producing said lipase inhibitors.

A further object of the present invention is to provide foods andbeverages that have the lipase inhibitors of the invention incorporatedtherein.

Yet another object of the present invention is to provide an antioxidantthat can prevent a variety of conditions resulting from active oxygenincluding, for example, life-style related diseases such ashypertension, diabetes and hyperlipemia, cardiac diseases such asarteriosclerosis, and aging and cancer.

A still further object of the present invention is to provide a lipaseinhibitor and/or an antioxidant, an effective amount of which can becorrectly incorporated in foods or beverages.

Means for Solving the Problems

Epigallocathechin Dimers and Trimers

As means for attaining those objects, the present inventors foundingredients in oolong tea that inhibited pancreatic lipase essential tofat absorption. The present inventors evaluated the lipase inhibitoryactivity of the various polyphenols present in oolong tea and found thatan epigallocatechin dimer of the following structure in whichepigallocatechin units are polymerized via a methylene group thatbridges their chroman rings at 6- or 8-position:

wherein R₁ and R₂ are each independently H or a galloyl group, had highinhibitory activity.

Preferred dimers of the present invention are compounds represented bythe following formulas:

which are oolong homobisflavan A(1), oolong homobisflavan B(2),monodesgalloyl oolong homobisflavan A(5), and didesgalloyl oolonghomobisflavan B(6).

According to the present invention, a novel, synthesizedepigallocatechin dimer, named oolong homobisflavan C(3):

also had high lipase inhibitory activity.

Also according to the present invention, an epigallocatechin trimer ofthe following structure in which epigallocatechin units are polymerizedvia methylene groups that bridge their chroman rings at 6- and/or8-position:

wherein R₃, R₄ and R₅ are each independently H or a galloyl group, alsohad strong lipase inhibitory activity.

A preferred epigallocatechin trimer is a compound represented by thefollowing formula:

which is named as compound (4).Production of Epigallocatechin Dimers and Trimers

The epigallocatechin dimers of the present invention can mostly besynthesized or purified for recovery by methods well known to skilledartisans. For example, oolong homobisflavan A(1) and B(2) which are bothepigallocatechin dimers can be synthesized by the method described inChem. Pharm. Bull 37(12), 3255-3563 (1989), whereas monodesgalloyloolong homobisflavan A(5) and didesgalloyl oolong homobisflavan B(6) canbe recovered from tea leaves by the method described in Chem. Pharm.Bull 37(12), 3255-3563 (1989).

The trimers of the present invention are produced by the followingprocedure. Briefly, they can be produced by a process including the stepof reacting flavan-3-ols such as epigallocatechin-3-O-gallate (7) withformaldehyde in a solvent such as methanol or ethanol, preferablymethanol, in the presence of an acid such as hydrochloric acid orsulfuric acid, preferably 0.01N hydrochloric acid. The reactiontemperature is not limited to any particular value as long as thestarting materials and the product are stable but it is preferred tocarry out the reaction at room temperature. Alternatively, the trimerscan be produced by dissolving flavan-3-ol such asepigallocatechin-3-O-gallate (7) in dimethyl sulfoxide (DMSO) andheating the solution. The heating reaction conditions are not limited inany particular way as long as the starting materials and the product arestable but the reaction is preferably carried out at 150-200° C., morepreferably at 180° C., for a period that is preferably between 1 and 30minutes, more preferably for 15 minutes. The concentration offormaldehyde in the reaction solution is preferably 3-37 w/v %. Theresulting product may optionally be esterified, hydrolyzed or otherwisetreated to make derived forms.

The methods described above for producing the trimers are also suitablefor producing the dimers. For example, as will be later described inExample 3, some dimers including novel homobisflavan C(3) were producedby those methods.

Lipase Inhibitory Activity

The compounds of the present invention have lipase inhibitory activity,in particular, pancreatic lipase inhibitory activity.

Lipase inhibitory activity can be measured by any one of the lipaseactivity assays that are described in the prior applications mentionedin the Background Art. As for inhibitory activity against pancreaticlipase, an oleic ester of fluorescent 4-methylumbelliferone may be usedas a substrate to measure the fluorescence from 4-methylumbelliferonethat is formed by reaction with the lipase. The amount of a sample thatprovides 50% inhibition of lipase activity (IC₅₀) may be substituted toexpress lipase inhibitory activity in accordance with the usual method.

As will be shown in Example 1, compounds of the present invention hadvery low IC₅₀ values against pancreatic lipase as compared with theknown lipase inhibitor epigallocatechin-3-O-gallate (7).

Therefore, the lipase inhibitors of the present invention can be used insmaller amounts than the heretofore known lipase inhibitors of naturalorigin and can yet suppress body absorption of meal-derived fat, therebysuppressing the elevation of blood neutral fat and/or preventingobesity. Since the compounds of the present invention are of naturalorigin, they feature high safety levels and are suitable for dailyand/or prolonged intake so that they can exhibit the intended efficacy.In addition, the compounds of the present invention which originate fromoolong tea have the advantage of suiting most consumers' taste.

Antioxidant Activity

The present inventors also found that the dimers and trimers of thepresent invention had the activity of scavenging superoxide anionradicals.

Superoxide anion radicals (O₂ ⁻) are a kind of active oxygen formedwithin the living body, where the radicals not only exhibit sterilizingaction but also induce an indiscriminate, strong oxidizing reaction.This effect is believed to cause conditions such as aging andcanceration in the living body, typically through the peroxidation ofunsaturated fatty acids in cell membranes (see, for example, NANZANDO'SMEDICAL DICTIONARY, 18th ed., p. 329, published Jan. 16, 1998). Inaddition, a peroxidation reaction of unsaturated fatty acids in foodwill lead to its deterioration and may even be involved in the emissionof off-odor from the food. Therefore, the compounds of the presentinvention which can scavenge superoxide anion radicals have beneficialcharacteristics in that they can prevent a variety of conditionsresulting from active oxygen including, for example, life-style relateddiseases such as hypertension, diabetes and hyperlipemia, cardiacdiseases such as arteriosclerosis, and aging and cancer.

The superoxide anion radical scavenging activity was measured by themethod to be described in Example 5. Briefly, O₂ ⁻ was generated bymeans of hypoxanthine-xanthine oxidase reaction and5,5-dimethyl-1-pyrrolin-N-oxide was used as a trapping agent to performmeasurement by ESR. Measurements were conducted in the presence orabsence of compounds of the present invention and the results wereexpressed as IC₅₀ or the amount of a sample that provided 50%suppression of O₂ ⁻ generation.

According to Example 5, compounds of the present invention had very lowIC₅₀ values as compared with compound (7), a known superoxide anionradical scavenger.

Therefore, the compounds of the present invention can be used in smalleramounts than the conventional superoxide anion radical scavengers ofnatural origin and can yet prevent a variety of conditions resultingfrom active oxygen including, for example, life-style related diseasessuch as hypertension, diabetes and hyperlipemia, cardiac diseases suchas arteriosclerosis, and aging and cancer. In addition, since thecompounds of the present invention are of natural origin, they featurehigh safety levels and can be ingested over a prolonged period toexhibit the intended efficacy.

Food and Beverage Having Lipase Inhibitor and/or AntioxidantIncorporated Therein

The epigallocatechin dimers and/or trimers of the present invention maybe incorporated in tea in order to potentiate the polyphenols in it,thereby producing foods and beverages that have not only the action ofreducing neutral fat and preventing peroxidation of lipids, aging andobesity, but also the action of preventing a variety of conditionsresulting from active oxygen including, for example, life-style relateddiseases such as hypertension, diabetes and hyperlipemia, cardiacdiseases such as arteriosclerosis, and aging and cancer.

Examples of beverages in which the compounds of the present inventionmay be incorporated include soft drinks, tea beverages, liquid tonics,health drinks, nutrition supply drinks, sports drinks and carbonateddrinks (including liquid concentrates and preparatory powders for thesebeverages), and exemplary foods in which the compounds may beincorporated include gums, candies, jellies, confectioneries in tabletform, health foods, nutrition supply foods, and dietary supplements.

Pharmaceutical Composition Comprising Lipase Inhibitor and/orAntioxidant

The epigallocatechin dimers and/or trimers of the present invention maybe used to produce pharmaceuticals that have not only such actions asthe one of reducing triglycerides, but also the action of preventing avariety of conditions resulting from active oxygen including, forexample, life-style related diseases such as hypertension, diabetes andhyperlipemia, cardiac diseases such as arteriosclerosis, and aging andcancer.

If the compounds of the present invention are to be used as medicines,they are provided in the form of powder, granule, tablet, capsule,solution, injection, dermal solution, emulsion, ointment, etc.

Cosmetics Having Lipase Inhibitor and/or Antioxidant IncorporatedTherein

The epigallocatechin dimers and/or trimers of the present invention maybe used to produce cosmetics that have various actions including theprevention of lipid peroxidation, retardation of aging, and skinwhitening.

Cosmetics in which the compounds of the present invention may beincorporated are facial, skin and hair creams, lotions, gels, mousse,shampoo, rinse, etc.

The compounds of the present invention, which may be synthesizedproducts or purified extraction, may independently be used infoods/beverages, pharmaceutical compositions or cosmetics;alternatively, a mixture comprising more than one compound of thepresent invention may be added to foods or beverages.

Therefore, if an extract from a starting material such as tea leavescontains more than one compound of the present invention, the extractedmixture may be employed as the lipase inhibitor of the present inventionwithout separating the individual components.

Advantages of the Invention

By adding whichever of oolong tea's polyphenols or epigallocatechindimers and/or trimers that is a component of the higher lipaseinhibitory activity, one can provide beverages that retain the flavor oftea, suit many consumers' taste, and serve the purposes of preventingobesity and promoting health.

The compounds as well as foods and beverages of the present inventionalso have antioxidant activity and contribute to preventing lipidperoxidation and ageing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structural formulas of the samples subjectedto the evaluation of their lipase inhibitory activity and antioxidantactivity;

FIG. 2 is a ¹H-NMR spectrum of oolong homobisflavan C(3);

FIG. 3 is a ¹³C-NMR spectrum of oolong homobisflavan C(3);

FIG. 4 is a ¹H-NMR spectrum of trimer (4); and

FIG. 5 is a ¹³C-NMR spectrum of trimer (4).

The present invention is described more specifically with reference tothe following examples which are by no means intended to limit the scopeof the invention.

EXAMPLE 1 Measurement of Lipase Inhibitory Activity

For lipase activity measurement, an oleic ester of fluorescent4-methylumbelliferone (4-UMO) was used as a substrate and thefluorescence from 4-methylumbelliferone formed by enzyme reaction wasmeasured.

The buffer used in the measurement was 13 mM tris-HCl (pH 8.0)containing 150 mM NaCl and 1.36 mM CaCl₂. The substrate 4-UMO (productof Sigma-Aldrich Co.) was prepared as 0.1 M solution in DMSO and thendiluted 1.000-fold with the buffer. The lipase subjected to enzymemeasurement was swine pancreatic lipase (product of Sigma) that had beendiluted to a concentration of 400 U/ml with the buffer.

To start the enzyme reaction, 50 μl of buffered 4-UMO solution and 25 μlof distilled water (or an aqueous solution of sample) were loaded andmixed on a 96-well microplate and 25 μl of the buffered lipase solutionwas then added. After 30-minute reaction, 100 μl of 0.1 M citratebuffered solution (pH 4.2) was added to quench the reaction and thefluorescence from 4-methylumbelliferone (excitation wavelength: 355 nm;fluorescence wavelength: 460 nm), which was generated by the reaction,was measured with a fluorescence plate reader (Fluoroskan Asent CF:product of Labsystems).

The inhibitory activity of the test sample was determined as its amountthat would provide 50% inhibition of lipase activity (IC₅₀ in μl)compared to the activity of the control (distilled water).

EXAMPLE 2 Samples of Measurement

Oolong homobisflavan A(1) and oolong homobisflavan B(2) were synthesizedin accordance with the article Chem. Pharm. Bull 37(12), 3255-3563(1989). Monodesgalloyl oolong homobisflavan A(5) and didesgalloyl oolonghomobisflavan B(6) were extracted and purified from tea leaves by themethod described in Chem. Pharm. Bull 37(12), 3255-3563 (1989).Alternatively, they can be obtained by hydrolysis of oolonghomobisflavan A(1) and oolong homobisflavan B(2) with tannase. Novelcompounds, oolong homobisflavan C(3) and trimer (4), were synthesizedand purified by the methods of Examples 3 and 4 and thereafterdetermined by instrumental analysis to have the structures shown inFIG. 1. Incidentally, (−)-epigallocatechin-3-O-gallate (EGCG, compound7) was purchased from Wako Pure Chemical Industries, Ltd.

EXAMPLE 3 Synthesis and Purification of Oolong Homobisflavans (OHBFs)

A. Synthesis:

A hundred milligrams of (−)-epigallocatechin-3-O-gallate (Wako PureChemical Industries, Ltd.) was dissolved in 2 ml of methanol containing0.01N HCl and 18.5% formaldehyde and the solution was stirred at roomtemperature for 1 hour. After the end of the reaction, the mixture waspurified by high-performance liquid chromatography.

B. Conditions of Preparative HPLC:

Column: Develosil C30-UG-5 (2 cmφ×25 cm; Nomura Chemical Co., Ltd.)

Mobile phase: A, 0.1% TFA/H₂O; B, 90% CH₃CN, 0.1% TFA/H₂O; 6 ml/min

Gradient program: B 10%→40% (0-40 min); B 40% iso (40-60 min)

Detection: A 280 nm

Preparative HPLC was performed under the conditions specified above,yielding OHBF-A, OHBF-B, OHBF-C and the trimer in respective amounts of24.2 mg, 17.2 mg, 5.6 mg, and 13.8 mg. The individual compounds weresubjected to MS by micromass Q-TOF in a positive mode, whereupon [M+H]⁺ion peaks were detected at m/z 929, 929, 929 and 1399 for OHBF-A,OHBF-B, OHBF-C and the trimer, respectively. The spectral data forOHBF-A and OHBF-B agreed with those shown in Chem. Pharm. Bull 37(12),3255-3563 (1989), determining that those compounds had the structuresrepresented by formulas 1 and 2 in FIG. 1. As regards OHBF-C and thetrimer, NMR measurements for ¹H-NMR, ¹³C-NMR, ¹H{¹³C}-HSQC,¹H{¹³C}-HMBC, TOCSY and DQF-COSY were performed with DMX-750spectrometer (BRUKER BIOSPIN) to show that those compounds had thestructures represented by formulas 3 and 4 in FIG. 1. The ¹H-NMR and¹³C-NMR spectra of OHBF-C are shown in FIGS. 2 and 3, whereas the ¹H-NMRand ¹³C-NMR spectra of the trimer are shown in FIGS. 4 and 5.

EXAMPLE 4 Lipase Inhibitory Activities of Oolong Homobisflavans andtheir Trimer

The lipase inhibitory activities of six compounds (dimers of OHBFs andtheir trimer), as well as epigallocatechin gallate (EGCG) were measuredin accordance with Example 1 and the results are shown in Table 1. Thechemical structural formulas of the compounds evaluated in Example 4 arelisted in FIG. 1.

Dimers of flavan-3-ol having a galloyl group attached to the chromanring at 3-position via an ester bond, for example, oolong homobisflavanA, B and C, as well as the trimer showed potent lipase inhibitoryactivity compared with EGCG, so it was suggested that the dimers andtrimer in which EGCGs are polymerized would be useful as lipaseinhibitors.

Among those compounds, oolong homobisflavan A and B having particularlystrong lipase inhibitory activity had been verified to be contained inoolong tea.

TABLE 1 Lipase Inhibitory Activities of Various Polyphenols IC₅₀ (μM)Oolong homovisflavan A(1) 0.049 Oolong homobisflavan B(2) 0.108 Oolonghomobisflavan C(3) 0.097 Trimer (4) 0.129 Monodesgalloyl oolonghomobisflavan A(5) 0.271 Didesgalloyl oolong homobisflavan B(6) 2.083Epigallocathechin 3-O-gallate (7) 0.349

EXAMPLE 5 Active Oxygen Scavenging Activity

Evaluation was made of the activity for scavenging superoxide anionradicals generated by hipoxanthine-xanthine oxidase reaction, a kind ofactive oxygen.

Method:

After preparing (1) 2 mM hypoxanthine solution (dissolved in 0.1 Mphosphate buffered solution at pH 7.4) and (2) 5.5 mMdiethylenetriamine-pentaacetic acid (dissolved in 0.1 M phosphatebuffered solution at pH 7.4), 5.0 ml of the solution (1), 3.5 ml of thesolution (2), and 1.5 ml of 5,5-dimethyl-1-pyrrolin-N-oxide (DMPO,product of LABOTECH) were mixed to make reagent 1. A 50-μl portion ofreagent 1 was put in a 1.5-ml microtube and incubated at 37° C. for 4minutes. To the incubated reagent 1, a methanol solution of the samplewas added in an amount of 20 μl and then 50 μl of xanthine oxidase (0.4units/ml) was added, followed by stirring for 10 seconds. The reactionmixture was injected into a hematocrit tube, which was set in an ESRapparatus and sweeping in a magnetic field was started 60 seconds afteraddition of xanthine oxidase. The conditions of ESR measurement were asfollows: Power, 4 mW; C. Field, 335.5 mT, SwWid (±), 5 mT; SwTime, 1min; ModWid, 0.1 mT; Amp, 160; TimeC, 0.1 sec; Temperature, 20° C.

Among the ESR signals from DMPO-OOH, the signal of the lowest fieldstrength was compared in height to the internal standard Mn²⁺ signal andthe ratio (S/M) was assumed to represent the amount of O₂ ⁻ and the O₂ ⁻scavenging activity was calculated by the following formula:O₂ ⁻ scavenging activity (%)=100−{100×(S/M in the presence of thesample)/(S/M in the absence of the sample)}Results:

To evaluate the ability of oolong homobisflavans A, B and C (dimers), aswell as the trimer to scavenge superoxide anion radicals (O₂ ⁻), theirconcentrations capable of scavenging 50% O₂ ⁻ (IC₅₀) were calculated inμM. The control was epigallocatechin gallate (EGCG). Compared to EGCG,the oolong homobisflavans (dimers and trimer) were found to havestronger O₂ ⁻ scavenging activity.

TABLE 2 Superoxide Radical Scavenging Activity Sample M.W. IC₅₀ (μM)Oolong homobisflavan A(1) 928 7.9 Oolong homobisflavan B(2) 928 8.1Oolong homobisflavan C(3) 928 6.2 Trimer (4) 1398 10.1 EGCG (7) 458 14.0

1. A method for inhibiting lipase activity comprising administering a composition consisting essentially of an epigallocatechin dimer in which epigallocatechin units are polymerized via a methylene group that bridges their chroman rings at 6- or 8-position:

wherein R₁ and R₂ are each independently H or a galloyl group, and/or an epigallocatechin trimer in which epigallocatechin units are polymerized via methylene groups that bridge their chroman rings at 6- and/or 8-position:

wherein R₃, R₄ and R₅ are each independently H or a galloyl group.
 2. The method of claim 1, wherein the epigallocatechin dimer in which epigallocatechin units are polymerized via a methylene group that bridges their chroman rings at 6- or 8-position is oolong homobisflavan A of the formula:

R=galloyl group, oolong homobisflavan B of the formula:

R1=R2=galloyl group, monodesgalloyl oolong homobisflavan A of the formula:

R═H, didesgalloyl oolong homobisflavan B of the formula:

R1=R2=H, or oolong homobisflavan c of the formula:

and an epigallocatechin trimer in which epigallocatechin units are polymerized via methylene groups that bridge their chroman rings at 6- and/or 8-positions is a compound of the formula:


3. The method of claim 1, wherein the composition is a pharmaceutical or food composition.
 4. The method of claim 1, wherein the composition is a drink or food.
 5. The method of claim 1, wherein the composition is a tea beverage.
 6. A method for suppressing body absorption of meal-derived fat, for suppressing the elevation of blood neutral fat, or for suppressing obesity, comprising administering a composition consisting essentially of an epigallocatechin dimer in which epigallocatechin units are polymerized via a methylene group that bridges their chroman rings at 6- or 8-position:

wherein R₁ and R₂ are each independently H or a galloyl group, and/or an epigallocatechin trimer in which epigallocatechin units are polymerized via methylene groups that bridge their chroman rings at 6- and/or 8-position:

wherein R₃, R₄ and R₅ are each independently H or a galloyl group.
 7. The method of claim 6, wherein the epigallocatechin dimer in which epigallocatechin units are polymerized via a methylene group that bridges their chroman rings at 6- or 8-position is oolong homobisflavan A of the formula:

R=galloyl group, oolong homobisflavan B of the formula:

R1=R2=galloyl group, monodesgalloyl oolong homobisflavan A of the formula:

R═H, didesgalloyl oolong homobisflavan B of the formula:

R1=R2=H, or oolong homobisflavan c of the formula:

and an epigallocatechin trimer in which epigallocatechin units are polymerized via methylene groups that bridge their chroman rings at 6- and/or 8-positions is a compound of the formula:


8. A method for inhibiting lipase activity comprising administering to a mammal at least one of the compounds selected from the group consisting of an epigallocatechin dimer in which epigallocatechin units are polymerized via a methylene group that bridges their chroman rings at 6- or 8-position:

wherein R₁ and R₂ are each independently H or a galloyl group, and an epigallocatechin trimer in which epigallocatechin units are polymerized via methylene groups that bridge their chroman rings at 6- and/or 8-position:

wherein R₃, R₄ and R₅ are each independently H or a galloyl group.
 9. The method of claim 8, wherein the epigallocatechin dimer is oolong homobisflavan A of the formula:

R=galloyl group, oolong homobisflavan B of the formula:

R1=R2=galloyl group, monodesgalloyl oolong homobisflavan A of the formula:

R═H, didesgalloyl oolong homobisflavan B of the formula:

R1=R2=H, or oolong homobisflavan c of the formula:

and an epigallocatechin trimer is a compound of the formula:


10. The method of claim 8 or 9, wherein at least one of the compounds is included in a food, a beverage, or a pharmaceutical composition.
 11. The method of claim 10, wherein the food or beverage is selected from the group consisting of tea drinks, soft drinks, and health foods.
 12. A method for suppressing body absorption of meal-derived fat, for suppressing the elevation of blood neutral fat, or for suppressing obesity, comprising administering to a mammal at least one of the compounds selected from the group consisting of an epigallocatechin dimer in which epigallocatechin units are polymerized via a methylene group that bridges their chroman rings at 6- or 8-position:

wherein R₁ and R₂ are each independently H or a galloyl group, and an epigallocatechin trimer in which epigallocatechin units are polymerized via methylene groups that bridge their chroman rings at 6- and/or 8-position:

wherein R₃, R₄ and R₅ are each independently H or a galloyl group.
 13. The method of claim 12, wherein the epigallocatechin dimer is oolong homobisflavan A of the formula:

R=galloyl group, oolong homobisflavan B of the formula:

R1=R2=galloyl group, monodesgalloyl oolong homobisflavan A of the formula:

R═H, didesgalloyl oolong homobisflavan B of the formula:

R1=R2=H, or oolong homobisflavan c of the formula:

and an epigallocatechin trimer is a compound of the formula:


14. The method of claim 12 or 13, wherein at least one of the compounds is included in a food, a beverage, or a pharmaceutical composition.
 15. The method of claim 14, wherein the food or beverage is selected from the group consisting of tea drinks, soft drinks, and health foods. 